JP5024291B2 - Fluorescent semiconductor fine particles, method for producing the same, fluorescent labeling agent for biological material using the same, and bioimaging method using the same - Google Patents
Fluorescent semiconductor fine particles, method for producing the same, fluorescent labeling agent for biological material using the same, and bioimaging method using the same Download PDFInfo
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- JP5024291B2 JP5024291B2 JP2008534278A JP2008534278A JP5024291B2 JP 5024291 B2 JP5024291 B2 JP 5024291B2 JP 2008534278 A JP2008534278 A JP 2008534278A JP 2008534278 A JP2008534278 A JP 2008534278A JP 5024291 B2 JP5024291 B2 JP 5024291B2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6489—Photoluminescence of semiconductors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
- Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
- Y10T428/2991—Coated
Description
本発明は、蛍光半導体微粒子、その製造方法、それを用いた生体物質蛍光標識剤及びそれを用いたバイオイメージング法に関する。 The present invention relates to a fluorescent semiconductor fine particle, a production method thereof, a biological material fluorescent labeling agent using the same, and a bioimaging method using the same.
より詳しくは、分散安定であり、粒径分布の良好な蛍光半導体微粒子とその製造方法に関する。更には、細胞の動態解析を行う生物学や免疫解析分野における動態イメージングに有効な生体物質蛍光標識剤及びそれを用いたバイオイメージング法に関する。 More specifically, the present invention relates to a fluorescent semiconductor fine particle that is stable in dispersion and has a good particle size distribution, and a method for producing the same. Further, the present invention relates to a biological substance fluorescent labeling agent effective for dynamic imaging in the field of biological analysis and immunological analysis for analyzing cell dynamics and a bioimaging method using the same.
近年、粒径により蛍光波長を制御可能である半導体ナノ粒子の研究が盛んに行われている。半導体ナノ粒子は蛍光波長の制御性、高退光性、表面修飾の自由度の高さを有することから生体内外の蛍光マーカーとして応用され、研究されている。 In recent years, research on semiconductor nanoparticles in which the fluorescence wavelength can be controlled by the particle size has been actively conducted. Semiconductor nanoparticles are applied and studied as fluorescent markers inside and outside the living body because they have controllability of fluorescence wavelength, high light-retarding property, and high degree of freedom of surface modification.
特に最近、バルクの生体認識で定性的なアッセイから、より分子レベルで動態を解析することにより生細胞内生体分子の反応機構を掴む基礎医学的研究や、疾病の原因となるウイルス・細菌の生態作用、薬の生体作用を解明しようとするバイオイメージングの研究が盛んであり、特に分子イメージングに代表されるように蛍光標識剤１分子又は数分子に対し標識される生体物質（細胞内の核、小胞体、ゴルジ体、蛋白、抗体、ＤＮＡ、ＲＮＡ、）１分子が結合し、所定の励起光を照射することによる発光を検知することで、これまで得られなかった生体情報（ＤＮＡ転写ｍＲＮＡ・蛋白形成に至る動態や細胞アポトーシス動態等）が得られようになっている。この場合、標的となる生体分子の生体内での本来の動態を追跡することが重要であり、そのため標的分子に吸着もしくは結合する標識物質は標的の動きを阻害しないものであることが望まれていた。また１分子の動態追跡であるがために、従来から１検体当りの検知する発光量が乏しく精度に乏しいことが指摘されており、更なる精度向上も望まれていた（例えば、特許文献１〜３参照）。 In particular, from recent qualitative assays in bulk biorecognition, basic medical research to grasp the reaction mechanism of biomolecules in living cells by analyzing dynamics at a more molecular level, and the ecology of viruses and bacteria that cause diseases Research on bioimaging to elucidate the biological action of drugs and drugs is thriving. In particular, as represented by molecular imaging, biological substances labeled with one or several fluorescent labeling agents (nuclear cells in cells, ER, Golgi, protein, antibody, DNA, RNA) molecules bind to each other and detect luminescence by irradiating with a predetermined excitation light. Dynamics leading to protein formation, cell apoptosis dynamics, etc.) have been obtained. In this case, it is important to track the original dynamics of the target biomolecule in the living body. Therefore, it is desired that the labeling substance that adsorbs or binds to the target molecule does not inhibit the movement of the target. It was. In addition, since it is a dynamic tracking of one molecule, it has been pointed out that the amount of luminescence detected per specimen is poor and the accuracy is low, and further improvement in accuracy has been desired (for example, Patent Documents 1 to 3). 3).
更に従来生体標識に使われてきた有機色素、蛍光タンパクにおいては発光量が乏しいことから検出性を上げる工夫（例えば、励起光強度の増大化、焦点を絞ることによる励起量の増加等の工夫）をしようとした場合に速やかな退色が生じ、検出性と耐久性のバランスがとれずにイメージングの精度が低いことが指摘され、特に分子イメージングにおいては難題となっていた。 In addition, organic dyes and fluorescent proteins that have been used for biolabeling in the past have been designed to increase the detectability due to the lack of light emission (for example, to increase the excitation light intensity, increase the excitation amount by focusing, etc.) When trying to do this, quick fading occurs, and it has been pointed out that the accuracy of imaging is low because the balance between detectability and durability is not balanced, and this has been a difficult problem especially in molecular imaging.
一方、生体適用の標識剤とするためには水溶液に分散安定であることが必要であり、粒径分布の良い粒子が分散したまま生体に供与されることが検出性に重要である。従来使用されてきた半導体微粒子（ＣｄＳｅ、ＣｄＴｅなど）はその比重などの性質上、溶液中に良好に分散するには多くの分散剤（活性剤類）を表面に付着させておく必要があり、またすぐに沈降してしまう問題があり分散液状態での合成供給に課題があった。分散を良好にするために分散剤を多く付着さシェルと粒子は大粒径化して標的分子の動態を阻害し、沈降し凝集体形成すると粗大粒子が増え粒径分布も劣化するために検出が一様でなくなり精度を著しくおとすこととなるという問題が指摘されていた。
本発明は、上記問題に鑑みてなされたものであり、その解決課題は、検出精度の高い動態イメージング性を実現する蛍光半導体微粒子、その製造方法、それを用いた生体物質蛍光標識剤及びバイオイメージング法を提供することである。 The present invention has been made in view of the above-mentioned problems, and the problem to be solved is a fluorescent semiconductor fine particle that realizes dynamic imaging with high detection accuracy, a method for producing the same, a biological substance fluorescent labeling agent using the same, and bioimaging Is to provide the law.
本発明に係る上記課題は下記の手段により解決される。 The above-mentioned problem according to the present invention is solved by the following means.
１．半導体微粒子からなるコア粒子と該コア粒子を被覆するシェル層とで構成されるコア／シェル構造を有する蛍光半導体微粒子であって、該コア粒子とシェル層の化学組成が相異し、該コア粒子の平均粒径が１〜１５ｎｍであり、比重が１．０〜３．０であり、かつ該コア／シェル構造を有する蛍光半導体微粒子の比重が０．８〜３．２であることを特徴とする蛍光半導体微粒子。 1. Fluorescent semiconductor fine particles having a core / shell structure composed of core particles composed of semiconductor fine particles and a shell layer covering the core particles, wherein the core particles and the shell layer have different chemical compositions, and the core particles The average particle diameter is 1 to 15 nm, the specific gravity is 1.0 to 3.0, and the specific gravity of the fluorescent semiconductor fine particles having the core / shell structure is 0.8 to 3.2. Fluorescent semiconductor fine particles.
２．前記シェル層の比重が、前記コア／シェル構造を有する蛍光半導体微粒子の比重が０．８〜３．２になるように、コア粒子とシェル層の体積比により調整されることを特徴とする前記１に記載の蛍光半導体微粒子。 2. The specific gravity of the shell layer is adjusted by the volume ratio of the core particles to the shell layer so that the specific gravity of the fluorescent semiconductor fine particles having the core / shell structure is 0.8 to 3.2. The fluorescent semiconductor fine particle according to 1.
３．前記１又は２に記載の蛍光半導体微粒子であって、前記コア粒子の平均粒径が１〜１０ｎｍであり、かつコア／シェル構造を有する蛍光半導体微粒子の平均粒径が３〜１５ｎｍであることを特徴とする蛍光半導体微粒子。 3. 3. The fluorescent semiconductor fine particles according to 1 or 2, wherein the average particle diameter of the core particles is 1 to 10 nm, and the average particle diameter of the fluorescent semiconductor fine particles having a core / shell structure is 3 to 15 nm. Featuring fluorescent semiconductor particles.
４．前記１〜３のいずれか一項に記載の蛍光半導体微粒子であって、前記コア粒子の平均粒径が１〜５ｎｍであり、かつコア／シェル構造を有する蛍光半導体微粒子の平均粒径が３〜１０ｎｍであることを特徴とする蛍光半導体微粒子。 4). The fluorescent semiconductor fine particles according to any one of 1 to 3, wherein the average particle diameter of the core particles is 1 to 5 nm, and the average particle diameter of the fluorescent semiconductor fine particles having a core / shell structure is 3 to 3. Fluorescent semiconductor fine particles characterized by being 10 nm.
５．前記１〜４のいずれか一項に記載の蛍光半導体微粒子の製造方法であって、液相法によりコア粒子又はシェル層を形成することを特徴とする蛍光半導体微粒子の製造方法。 5). 5. The method for producing a fluorescent semiconductor fine particle according to any one of 1 to 4, wherein the core particle or the shell layer is formed by a liquid phase method.
６．前記１〜４のいずれか一項に記載の蛍光半導体微粒子の表面上に生体物質に結合する官能基と該蛍光半導体微粒子の表面に結合する官能基をもつ表面修飾化合物を有することを特徴とする生体物質蛍光標識剤。 6). A surface modifying compound having a functional group that binds to a biological material and a functional group that binds to the surface of the fluorescent semiconductor fine particle on the surface of the fluorescent semiconductor fine particle according to any one of 1 to 4 above. Biological substance fluorescent labeling agent.
７．前記６に記載の生体物質蛍光標識剤であって、その平均粒径が１〜２０ｎｍであることを特徴とする生体物質蛍光標識剤。 7). 7. The biological material fluorescent labeling agent according to 6, wherein the average particle diameter is 1 to 20 nm.
８．前記６又は７に記載の生体物質蛍光標識剤であって、その平均粒径が１〜１０ｎｍであることを特徴とする生体物質蛍光標識剤。 8). 8. The biological material fluorescent labeling agent according to 6 or 7, wherein the biological particle fluorescent labeling agent has an average particle diameter of 1 to 10 nm.
９．前記６〜８のいずれか一項に記載の生体物質蛍光標識剤を用いて蛍光動態イメージングを行うことを特徴とするバイオイメージング法。 9. A bioimaging method comprising performing fluorescence dynamic imaging using the biological material fluorescent labeling agent according to any one of 6 to 8 above.
本発明の上記手段により、分散安定で粒径分布が良好で、検出精度の高い動態イメージング性を実現する蛍光半導体微粒子、その製造方法、それを用いた生体物質蛍光標識剤及びバイオイメージング法を提供することができる。 By the above means of the present invention, a fluorescent semiconductor fine particle that realizes dynamic imaging properties with stable dispersion, good particle size distribution, and high detection accuracy, a method for producing the same, a fluorescent substance biolabeling agent using the same, and a bioimaging method are provided. can do.
なお、本発明により、特に１分子イメージングの研究分野において高感度高精度測定法を提供することができる。 The present invention can provide a high-sensitivity and high-precision measurement method particularly in the field of single-molecule imaging research.
以下、本発明とその構成要素等について詳細な説明をする。 Hereinafter, the present invention and its components will be described in detail.
（蛍光半導体微粒子）
本発明の蛍光半導体微粒子は半導体微粒子からなるコア粒子と該コア粒子を被覆するシェル層とで構成されるコア／シェル構造を有する蛍光半導体微粒子であって、該コア粒子とシェル層の化学組成が相異し、該コア粒子の平均粒径が１〜１５ｎｍであり、比重が１．０〜３．０であり、かつ該コア／シェル構造を有する蛍光半導体微粒子の比重が０．８〜３．２であることを特徴とする。また、前記シェル層の比重が、前記コア／シェル構造を有する蛍光半導体微粒子の比重が０．８〜３．２になるように、コア粒子とシェル層の体積比により調整されることを特徴とする。(Fluorescent semiconductor fine particles)
The fluorescent semiconductor fine particles of the present invention are fluorescent semiconductor fine particles having a core / shell structure composed of core particles made of semiconductor fine particles and a shell layer covering the core particles, and the chemical composition of the core particles and the shell layer is The average particle diameter of the core particles is 1 to 15 nm, the specific gravity is 1.0 to 3.0, and the specific gravity of the fluorescent semiconductor fine particles having the core / shell structure is 0.8 to 3. 2 is a feature. The specific gravity of the shell layer is adjusted by the volume ratio of the core particles to the shell layer so that the specific gravity of the fluorescent semiconductor fine particles having the core / shell structure is 0.8 to 3.2. To do.
ここで、「比重」とは、一般的に表現される同じ体積での４℃の水に対する質量比をいう。 Here, “specific gravity” refers to a mass ratio with respect to water at 4 ° C. in the same volume that is generally expressed.
本発明に係るコア粒子の比重は、上記のように１．０〜３．０であることが必要であり、好ましくは１．０〜２．５である。 The specific gravity of the core particles according to the present invention needs to be 1.0 to 3.0 as described above, and is preferably 1.0 to 2.5.
本発明の本発明の蛍光半導体微粒子の平均粒径は、３〜１５ｎｍが好ましく、更に好ましくは３〜１０ｎｍである。 The average particle size of the fluorescent semiconductor fine particles of the present invention is preferably 3 to 15 nm, more preferably 3 to 10 nm.
後述する生体標識剤の粒径は、その効果を発現さシェルために１〜２０ｎｍであることが好ましい、そのため本発明の蛍光半導体微粒子の平均粒径は１〜１０ｎｍであることが特に好ましい。 The particle size of the biomarker described later is preferably 1 to 20 nm in order to exhibit the effect, and therefore the average particle size of the fluorescent semiconductor fine particles of the present invention is particularly preferably 1 to 10 nm.
ここで、「平均粒径」とは、レーザー散乱法により測定される累積５０％体積粒径をいう。 Here, the “average particle diameter” refers to a cumulative 50% volume particle diameter measured by a laser scattering method.
なお、シェル層の厚みもしくはコア／シェル構造蛍光半導体微粒子の検出法としてはＴＥＭ（透過型顕微鏡）を観察し、コア粒子とコア／シェル構造粒子を比較することによっても求めることができるし、各々の粒径測定によっても求めることができる。 The thickness of the shell layer or the core / shell structure fluorescent semiconductor fine particles can be detected by observing a TEM (transmission microscope) and comparing the core particles with the core / shell structure particles. It can also be determined by measuring the particle size of
〈コア粒子〉
本発明に係るコア粒子は、本発明のコア／シェル構造を有する蛍光半導体微粒子のコア部分を形成する構成要素であるが、上記比重の条件を満たすことができる半導体材料により構成されることを要する。なお、必要があればＧａなどのドープ材料を極微量含んでもよい。<Core particle>
The core particle according to the present invention is a component that forms the core portion of the fluorescent semiconductor fine particle having the core / shell structure of the present invention, but it is necessary to be composed of a semiconductor material that can satisfy the above specific gravity condition. . If necessary, a trace amount of a doping material such as Ga may be included.
コアに用いられる半導体材料としては、本発明に係る上記比重条件を満たす使用方法において、種々の半導体材料を用いることができる。具体例としては、例えば、ＭｇＳ、ＭｇＳｅ、ＭｇＴｅ、ＣａＳ、ＣａＳｅ、ＣａＴｅ、ＳｒＳ、ＳｒＳｅ、ＳｒＴｅ、ＢａＳ、ＢａＴｅ、ＺｎＳ、ＺｎＳｅ、ＺｎＴｅ、ＣｄＳ、ＣｄＳｅ、ＣｄＴｅ、ＧａＡｓ、ＧａＰ、ＧａＳｂ、ＩｎＧａＡｓ、ＩｎＰ、ＩｎＮ、ＩｎＳｂ、ＩｎＡｓ、ＡｌＡｓ、ＡｌＰ、ＡｌＳｂ、ＡｌＳ、ＰｂＳ、ＰｂＳｅ、Ｇｅ、Ｓｉ、又はこれらの混合物等が挙げられる。本発明において、特に好ましい半導体材料は、Ｓｉである。 As a semiconductor material used for the core, various semiconductor materials can be used in the usage method that satisfies the specific gravity condition according to the present invention. Specific examples include, for example, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaAs, GaP, GaSb, InGaAs, InP. InN, InSb, InAs, AlAs, AlP, AlSb, AlS, PbS, PbSe, Ge, Si, or a mixture thereof. In the present invention, a particularly preferable semiconductor material is Si.
なお、上記の半導体材料等を用いて本発明に係る半導体ナノ粒子を形成する際には、上記の比重についての条件を満たすよう混合比率等を調整することが必要である。 In addition, when forming the semiconductor nanoparticles according to the present invention using the above-described semiconductor material or the like, it is necessary to adjust the mixing ratio or the like so as to satisfy the above-described specific gravity conditions.
本発明に係るコアの平均粒径に関しては、発明の効果発現のために、１〜１５ｎｍであることを要する。なお、平均粒径を１〜１０ｎｍとすることにより小粒径の生体分子の標識及び検知が可能となり、更に、１〜５ｎｍであれば、十分に生体１分子に対する標識並びに動態イメージングが可能となる。従って、より好ましいのは１〜１０ｎｍ、特に好ましいのは１〜５ｎｍである。 The average particle diameter of the core according to the present invention is required to be 1 to 15 nm in order to achieve the effect of the invention. By setting the average particle size to 1 to 10 nm, it is possible to label and detect biomolecules having a small particle size. Further, if the average particle size is 1 to 5 nm, sufficient labeling and dynamic imaging can be performed for one biological molecule. . Therefore, 1 to 10 nm is more preferable, and 1 to 5 nm is particularly preferable.
なお、本発明に係るコアの「平均粒径」とは、レーザー散乱法により測定される累積５０％体積粒径をいう。 The “average particle diameter” of the core according to the present invention refers to a cumulative 50% volume particle diameter measured by a laser scattering method.
〈シェル層〉
本発明に係るシェル層は、本発明の蛍光半導体微粒子において、上記コア粒子を被覆する層であり、コア／シェル構造を形成するための構成層であるが、上記比重の条件を満たすことができる半導体材料により構成されることを要する。<Shell layer>
The shell layer according to the present invention is a layer that covers the core particle in the fluorescent semiconductor fine particles of the present invention and is a constituent layer for forming a core / shell structure, but can satisfy the specific gravity condition. It needs to be composed of a semiconductor material.
なお、本発明に係るシェル層は、コア粒子が部分的に露出して弊害を生じない限り、コア粒子の全表面を完全に被覆するものでなくてもよい。 Note that the shell layer according to the present invention may not completely cover the entire surface of the core particle as long as the core particle is not partially exposed to cause a harmful effect.
シェルに用いられる半導体材料としては、本発明に係る上記比重条件を満たす使用方法において、種々の半導体材料を用いることができる。具体例としては、例えば、ＺｎＯ、ＺｎＳ、ＺｎＳｅ、ＺｎＴｅ、ＣｄＯ、ＣｄＳ、ＣｄＳｅ、ＣｄＴｅ、ＭｇＳ、ＭｇＳｅ、ＧａＳ、ＧａＮ、ＧａＰ、ＧａＡｓ、ＧａＳｂ、ＩｎＡｓ、ＩｎＮ、ＩｎＰ、ＩｎＳｂ、ＡｌＡｓ、ＡｌＮ、ＡｌＰ、ＡｌＳｂ、又はこれらの混合物等が挙げられる。 As a semiconductor material used for the shell, various semiconductor materials can be used in the usage method that satisfies the specific gravity condition according to the present invention. Specific examples include, for example, ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaS, GaN, GaP, GaAs, GaSb, InAs, InN, InP, InSb, AlAs, AlN, AlP. , AlSb, or a mixture thereof.
なお、好ましいシェルの材料としては、半導性ナノ結晶コアより高いバンドギャップエネルギーを有する半導性材料が挙げられる。 A preferred shell material is a semiconductive material having a higher band gap energy than the semiconductive nanocrystal core.
半導性微粒子結晶コアより高いバンドギャップエネルギーを有することに加えて、シェルに適切な材料は、コア半導性ナノ結晶に関して、良好な伝導性および原子価バンドオフセットを有するべきである。従って、伝導性バンドは、コア半導性ナノ結晶の伝導性バンドよりも望ましくは高く、そして原子価バンドは、コア半導性ナノ結晶の原子価バンドよりも望ましくは低い。可視で（例えば、Ｓｉ、Ｇｅ、ＧａＰ、）または近赤外で（例えば、ＩｎＰ、ＩｎＮ、ＰｂＳ、ＰｂＳｅ）エネルギーを放出する半導性ナノ結晶コアについて、紫外線領域でバンドギャップエネルギーを有する材料が使用され得る。具体例としては、例えば、ＺｎＳ、ＧａＮおよびマグネシウムカルコゲニド（例えば、ＭｇＳ、ＭｇＳｅおよびＭｇＴｅ）が挙げられる。 In addition to having a higher band gap energy than the semiconducting particulate crystal core, a suitable material for the shell should have good conductivity and valence band offset with respect to the core semiconducting nanocrystal. Thus, the conduction band is desirably higher than the conduction band of the core semiconducting nanocrystal, and the valence band is desirably lower than the valence band of the core semiconducting nanocrystal. For semiconducting nanocrystal cores that emit energy in the visible (eg, Si, Ge, GaP,) or near infrared (eg, InP, InN, PbS, PbSe), materials with bandgap energy in the ultraviolet region are Can be used. Specific examples include ZnS, GaN, and magnesium chalcogenide (for example, MgS, MgSe, and MgTe).
近赤外で放出する半導性ナノ結晶コアについて、可視でバンドギャップエネルギーを有する材料もまた使用され得る。 For semiconducting nanocrystal cores emitting in the near infrared, materials with visible band gap energy can also be used.
本発明において、特に好ましい半導体材料は、ＳｉＯ2、ＺｎＳである。In the present invention, particularly preferred semiconductor materials are SiO 2 and ZnS.
〈蛍光半導体微粒子の製造方法〉
本発明の蛍光半導体微粒子の製造については、従来公知の種々の方法を用いることができる。<Method for producing fluorescent semiconductor fine particles>
Various conventionally known methods can be used for the production of the fluorescent semiconductor fine particles of the present invention.
液相法の製造方法としては、沈殿法である、共沈法、ゾルーゲル法、均一沈殿法、還元法などがある。そのほかに、逆ミシェル法、超臨界水熱合成法、などもナノ粒子を作製する上で優れた方法である（例えば、特開２００２−３２２４６８号、特開２００５−２３９７７５号、特開平１０−３１０７７０号、特開２０００−１０４０５８号公報等を参照。）。 Examples of the liquid phase production method include precipitation methods such as coprecipitation method, sol-gel method, uniform precipitation method, and reduction method. In addition, the reverse Michel method, the supercritical hydrothermal synthesis method, and the like are also excellent methods for producing nanoparticles (for example, JP-A No. 2002-322468, JP-A No. 2005-239775, JP-A No. 10-310770). No., JP 2000-104058 A, etc.).
気相法の製造方法としては、（１）対向する原料半導体を電極間で発生させた第一の高温プラズマによって蒸発させ、減圧雰囲気中において無電極放電で発生させた第二の高温プラズマ中に通過さシェル方法（例えば特開平６−２７９０１５号公報参照。）、（２）電気化学的エッチングによって、原料半導体からなる陽極からナノ粒子を分離・除去する方法（例えば特表２００３−５１５４５９号公報参照。）、レーザーアブレーション法（例えば特開２００４−３５６１６３号参照。）などが用いられる。また、原料ガスを低圧状態で気相反応させて、粒子を含む粉末を合成する方法も、好ましく用いられる。 As a manufacturing method of the vapor phase method, (1) the opposing raw material semiconductor is evaporated by the first high temperature plasma generated between the electrodes, and in the second high temperature plasma generated by electrodeless discharge in a reduced pressure atmosphere. Passed shell method (for example, see JP-A-6-279015), (2) A method for separating and removing nanoparticles from an anode made of a raw material semiconductor by electrochemical etching (for example, see JP-A-2003-515458) ), A laser ablation method (see, for example, Japanese Patent Application Laid-Open No. 2004-356163), and the like are used. A method of synthesizing a powder containing particles by reacting a raw material gas in a gas phase in a low pressure state is also preferably used.
本発明の蛍光半導体微粒子の製造方法としては、特に液相法による製造方法が好ましい。すなわち、本発明においては、蛍光半導体微粒子のコア及びシェルが上記比重になる組成を選択する必要があるため、液相法による場合には、粒子形成後の分散液中の粒子沈降がほとんど無く良好に分散していることにより、粒径分布も狭い目的粒径に沿う粒子を形成しやすいからである。 As the method for producing the fluorescent semiconductor fine particles of the present invention, a production method by a liquid phase method is particularly preferable. That is, in the present invention, since it is necessary to select a composition in which the core and shell of the fluorescent semiconductor fine particles have the above specific gravity, in the case of the liquid phase method, there is almost no particle sedimentation in the dispersion after the particle formation. This is because it is easy to form particles along a target particle diameter having a narrow particle size distribution.
〈蛍光半導体微粒子の表面修飾〉
本発明の蛍光半導体微粒子を生体適用の標識剤とするためには、当該蛍光半導体微粒子の表面を表面修飾化合物を用いて修飾することを要する。<Surface modification of fluorescent semiconductor fine particles>
In order to use the fluorescent semiconductor fine particles of the present invention as a labeling agent for biological application, it is necessary to modify the surface of the fluorescent semiconductor fine particles with a surface modifying compound.
表面修飾化合物としては、少なくとも１つの官能基と少なくとも１つの蛍光半導体微粒子に結合する基を有する化合物であることが好ましい。後者は疎水性の蛍光半導体微粒子に吸着できる基であり、他方は生体物質に親和性があり生体分子に結合する官能基である。互いの表面修飾化合物は互いをつなぐ各種のリンカーを使用してもよい。 The surface modifying compound is preferably a compound having at least one functional group and at least one group bonded to the fluorescent semiconductor fine particles. The latter is a group that can be adsorbed to hydrophobic fluorescent semiconductor fine particles, and the other is a functional group that has affinity for biological substances and binds to biomolecules. The surface modification compounds of each other may use various linkers that connect each other.
蛍光半導体微粒子に結合する基としては、前記のシェル層若しくはコア粒子を形成するための半導体材料に結合する官能基であれば良い。従って、シェル層若しくはコア粒子の組成に応じて好ましい官能基を選択することが好ましい。本発明においては、当該官能基として、特にチオール基が好ましい。 The group bonded to the fluorescent semiconductor fine particles may be a functional group bonded to the semiconductor material for forming the shell layer or core particle. Therefore, it is preferable to select a preferred functional group according to the composition of the shell layer or core particle. In the present invention, the functional group is particularly preferably a thiol group.
生体物質に親和的に結合する官能基としては、カルボキシル基、アミノ基、フォスフォン酸基、スルホン酸基などが挙げられる。 Examples of the functional group that binds to the biological substance with affinity include a carboxyl group, an amino group, a phosphonic acid group, and a sulfonic acid group.
なお、ここで、「生体物質」とは、細胞、ＤＮＡ、ＲＮＡ、オリゴヌクレオチド、蛋白質、抗体、抗原、小胞体、核、ゴルジ体等を指す。 Here, “biological substance” refers to cells, DNA, RNA, oligonucleotides, proteins, antibodies, antigens, endoplasmic reticulums, nuclei, Golgi bodies, and the like.
又、蛍光半導体微粒子に結合さシェル方法としては、表面修飾に適するｐＨに調整することによりメルカプト基を粒子に結合さシェルことができる。それぞれ他端にはアルデヒド基、アミノ基、カルボキシル基が導入され、生体のアミノ基、カルボキシル基とペプチド結合することができる。また、ＤＮＡ、オリゴヌクレオチドなどにアミノ基、アルデヒド基、カルボキシル基を導入しても同様に結合さシェルことができる。 Further, as a shell method bonded to the fluorescent semiconductor fine particles, a shell in which a mercapto group is bonded to the particles can be formed by adjusting the pH to be suitable for surface modification. An aldehyde group, an amino group, and a carboxyl group are introduced into the other end, respectively, and a peptide bond can be formed with a biological amino group or carboxyl group. Further, even when an amino group, an aldehyde group, or a carboxyl group is introduced into DNA, oligonucleotide, etc., a bonded shell can be formed in the same manner.
蛍光半導体微粒子の表面修飾のための具体的調製は、例えば、Ｄａｂｂｏｕｓｉら（１９９７）Ｊ．Ｐｈｙｓ．Ｃｈｅｍ．Ｂ１０１：９４６３、Ｈｉｎｅｓら（１９９６）Ｊ．Ｐｈｙｓ．Ｃｈｅｍ．１００：４６８−４７１、Ｐｅｎｇら（１９９７）Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１１９：７０１９−７０２９、及びＫｕｎｏら（１９９７）Ｊ．Ｐｈｙｓ．Ｃｈｅｍ．１０６：９８６９に記載されている方法に準拠して行うことができる。 Specific preparations for surface modification of fluorescent semiconductor particles are described, for example, in Dabbousi et al. (1997) J. MoI. Phys. Chem. B101: 9463, Hines et al. (1996) J. MoI. Phys. Chem. 100: 468-471, Peng et al. (1997) J. MoI. Am. Chem. Soc. 119: 7019-7029, and Kuno et al. (1997) J. MoI. Phys. Chem. 106: 9869.
（生体物質蛍光標識剤とそれを用いるバイオイメージング法）
本発明の蛍光半導体微粒子は、以下に説明する事由に基づき、生体物質蛍光標識剤に適応することができる。また、標的（追跡）物質を有する生細胞もしくは生体に本発明に係る生体物質蛍光標識剤を添加することで、標的物質と結合もしくは吸着し、該結合体もしくは吸着体に所定の波長の励起光を照射し、当該励起光に応じて蛍光半導体微粒子から発生する所定の波長の蛍光を検出することにより、上記標的（追跡）物質の蛍光動態イメージングを行うことができる。すなわち、本発明に係る生体物質蛍光標識剤は、バイオイメージング法（生体物質を構成する生体分子やその動的現象を可視化する技術手段）に利用することができる。(Biological substance fluorescent labeling agent and bioimaging method using it)
The fluorescent semiconductor fine particles of the present invention can be applied to a biological material fluorescent labeling agent based on the reasons described below. Further, by adding the biological material fluorescent labeling agent according to the present invention to a living cell or living body having a target (tracking) substance, it binds or adsorbs to the target substance, and the conjugate or adsorbent has excitation light having a predetermined wavelength. , And detecting fluorescence of a predetermined wavelength generated from the fluorescent semiconductor fine particles in response to the excitation light, fluorescence dynamic imaging of the target (tracking) substance can be performed. That is, the biological material fluorescent labeling agent according to the present invention can be used for a bioimaging method (technical means for visualizing biological molecules constituting the biological material and dynamic phenomena thereof).
以下、生体物質蛍光標識剤及び関連技術等について詳しく説明する。 Hereinafter, the biological material fluorescent labeling agent and related technologies will be described in detail.
本発明に係る表面修飾した蛍光半導体微粒子（以下「表面修飾半導体微粒子」ともいう。）は、表面修飾化合物の官能基により、結合対の第１のメンバーとして働く親和性分子に結合させ得る。例えば、表面修飾化合物の親水性構造部分内に存在するイオン化可能基は、親和性分子への結合手段を提供し得る。 The surface-modified fluorescent semiconductor fine particles (hereinafter also referred to as “surface-modified semiconductor fine particles”) according to the present invention can be bound to an affinity molecule that acts as a first member of a binding pair by a functional group of the surface modifying compound. For example, ionizable groups present in the hydrophilic structural portion of the surface modifying compound may provide a means for binding to the affinity molecule.
なお、親和性分子に分子および分子セグメントを結合する適切な方法については、例えば、Ｈｅｒｍａｎｓｏｎ、Ｂｉｏｃｏｎｊｕｇａｔｅ Ｔｅｃｈｎｉｑｕｅｓ（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，ＮＹ，１９９６）に記載されている。 Appropriate methods for binding molecules and molecular segments to affinity molecules are described, for example, in Hermanson, Bioconjugate Technologies (Academic Press, NY, 1996).
親和性分子によるこのような表面修飾半導体微粒子との「結合体」は、生体物質すなわち生物学的化合物および化学的化合物の存在および／または量、生物系、生物学的プロセスにおける相互作用、生物学的プロセスの変更あるいは生物学的化合物の構造における変化を検出するために使用され得る。すなわち、表面修飾半導体微粒子に結合した場合、親和性分子は、結合対の第２のメンバーとして働く生物学的標的と相互作用し、生物学的プロセスまたは応答を検出するか、あるいは生物学的分子またはプロセスを変化させ得る。 A “conjugate” with such surface-modified semiconductor microparticles by affinity molecules is the presence and / or amount of biological material, ie biological and chemical compounds, biological systems, interactions in biological processes, biology It can be used to detect alterations in chemical processes or changes in the structure of biological compounds. That is, when bound to a surface-modified semiconductor microparticle, the affinity molecule interacts with a biological target that serves as the second member of the binding pair to detect a biological process or response, or a biological molecule Or it can change the process.
好ましい態様としては、親和性分子および生物学的標的の相互作用は、特異的結合を伴い、そして共有結合、非共有結合、疎水性、親水性、ファンデルワールスまたは磁性の相互作用を伴い得る。更に、親和性分子は生物学的標的と物理的に相互作用させ得る。 In preferred embodiments, the interaction of the affinity molecule and the biological target involves specific binding and may involve covalent, non-covalent, hydrophobic, hydrophilic, van der Waals or magnetic interactions. Furthermore, affinity molecules can physically interact with biological targets.
表面修飾半導体微粒子に結合する親和性分子は、天然に存在し得るかまたは化学的に合成され得、そして所望の物理学的、化学的または生物学的特性を有することが選択され得る。 Affinity molecules that bind to the surface-modified semiconductor microparticles can be naturally occurring or chemically synthesized and can be selected to have the desired physical, chemical or biological properties.
このような特性としては、タンパク質、核酸、シグナル伝達分子、原核生物細胞または真核生物細胞、ウイルス、細胞内オルガネラおよび他の任意の生物学的化合物との共有結合および非共有結合等が挙げられるが、これらに制限されない。 Such properties include proteins, nucleic acids, signaling molecules, prokaryotic or eukaryotic cells, viruses, intracellular organelles, and any other biological compound, such as covalent and non-covalent bonds. However, it is not limited to these.
このような分子の他の特性としては、生物学的プロセス（例えば、細胞周期、血液凝固、細胞死、転写、翻訳、シグナル伝達、ＤＮＡ損傷またはＤＮＡ切断、ラジカル生成、ラジカル除去など）に影響を与える能力、および生物学的化合物の構造（例えば、架橋、タンパク質分解切断、ラジカル損傷など）を変化さシェル能力等挙げられるが、これらに制限されない。 Other properties of such molecules may affect biological processes such as cell cycle, blood clotting, cell death, transcription, translation, signal transduction, DNA damage or DNA cleavage, radical generation, radical removal, etc. The ability to impart, and the ability to change the structure of a biological compound (eg, cross-linking, proteolytic cleavage, radical damage, etc.), but is not limited to such.
好ましい実施形態において、表面修飾半導体微粒子結合体は、調整可能な波長で光を放射する半導体微粒子を含み、そして核酸に結合される。当該結合は、直接的または間接的であり得る。核酸は、任意のリボ核酸、デオキシリボ核酸、ジデオキシリボ核酸または任意の誘導体およびその組み合わせであり得る。核酸はまた、任意の長さのオリゴヌクレオチドであり得る。このオリゴヌクレオチドは、一本鎖、二本鎖、三本鎖またはより高位の立体配置（例えば、ホリデー結合、環状一本鎖ＤＮＡ、環状二本鎖ＤＮＡ、ＤＮＡ立方体（Ｓｅｅｍａｎ（１９９８）Ａｎｎ．Ｒｅｖ．Ｂｉｏｐｈｙｓ．Ｂｉｏｍｏｌ．Ｓｔｒｕｃｔ．２７：２２５２４８を参照のこと））であり得る。 In a preferred embodiment, the surface-modified semiconductor microparticle conjugate comprises semiconductor microparticles that emit light at a tunable wavelength and are bound to a nucleic acid. The binding can be direct or indirect. The nucleic acid can be any ribonucleic acid, deoxyribonucleic acid, dideoxyribonucleic acid or any derivative and combinations thereof. The nucleic acid can also be an oligonucleotide of any length. This oligonucleotide is a single-stranded, double-stranded, triple-stranded or higher configuration (eg, holiday binding, circular single-stranded DNA, circular double-stranded DNA, DNA cube (Seeman (1998) Ann. Rev. Biophys.Biomol.Struct.27: 225248))).
本発明に係る半導体微粒子結合体のとりわけ好ましい使用態様は、以下のような核酸の検出および／または定量である：（ａ）ウイルス核酸；（ｂ）細菌核酸；および（ｃ）目的の多くのヒトの配列（例えば、単鎖ヌクレオチド多型）。本発明の範囲を制限することなしに、蛍光半導体微粒子結合体は、個々のヌクレオチド、デオキシヌクレオチド、ジデオキシヌクレオチドまたは任意の誘導体およびその組み合わせに結合する蛍光半導体微粒子を含み得、そしてＤＮＡ重合反応（例えば、ＤＮＡ配列決定、ＤＮＡへのＲＮＡの逆転写およびポリメラーゼ連鎖反応（ＰＣＲ））において使用される。 A particularly preferred mode of use of the semiconductor particulate conjugate according to the present invention is the detection and / or quantification of nucleic acids as follows: (a) viral nucleic acids; (b) bacterial nucleic acids; and (c) many humans of interest. Sequences (eg, single-stranded nucleotide polymorphisms). Without limiting the scope of the present invention, the fluorescent semiconductor microparticle conjugate can include fluorescent semiconductor microparticles that bind to individual nucleotides, deoxynucleotides, dideoxynucleotides or any derivative and combinations thereof, and a DNA polymerization reaction (eg, , DNA sequencing, reverse transcription of RNA into DNA and polymerase chain reaction (PCR)).
ヌクレオチドとしてはまた、一リン酸塩、二リン酸塩および三リン酸塩ならびに環状誘導体（例えば、環状アデニン一リン酸（ｃＡＭＰ））が挙げられる。 Nucleotides also include monophosphates, diphosphates and triphosphates and cyclic derivatives such as cyclic adenine monophosphate (cAMP).
核酸に結合した蛍光半導体微粒子の他の使用態様としては、蛍光インサイチュハイブリダイゼーション（ＦＩＳＨ）が含まれる。この好ましい実施形態において、蛍光半導体微粒子はインビボで特異的な配列にハイブリダイズするように設計されたオリゴヌクレオチドに結合される。ハイブリダイゼーションに関して、蛍光半導体微粒子タグは、細胞において所望のＤＮＡ配列の位置を可視化するために使用される。例えば、そのＤＮＡ配列が部分的または完全に既知である遺伝子の細胞内局在は、ＦＩＳＨを用いて決定され得る。 Other uses of fluorescent semiconductor fine particles bound to nucleic acids include fluorescent in situ hybridization (FISH). In this preferred embodiment, the fluorescent semiconductor microparticles are bound to an oligonucleotide designed to hybridize to a specific sequence in vivo. For hybridization, fluorescent semiconductor particulate tags are used to visualize the location of the desired DNA sequence in the cell. For example, the subcellular localization of a gene whose DNA sequence is partially or fully known can be determined using FISH.
その配列が部分的または完全に既知である任意のＤＮＡまたはＲＮＡは、ＦＩＳＨを用いて視覚的に標識され得る。例えば、本発明の範囲を制限することなしに、メッセンジャーＲＮＡ（ｍＲＮＡ）、ＤＮＡテロメア、他の高反復ＤＮＡ配列および他のコードされていないＤＮＡ配列がＦＩＳＨにより標的化され得る。 Any DNA or RNA whose sequence is partially or fully known can be visually labeled using FISH. For example, messenger RNA (mRNA), DNA telomeres, other highly repetitive DNA sequences and other non-encoded DNA sequences can be targeted by FISH without limiting the scope of the invention.
蛍光半導体微粒子結合体はまた、生物学的化合物（例えば、酵素、酵素基質、酵素インヒビター、細胞内オルガネラ、脂質、リン脂質、脂肪酸、ステロール、細胞膜、シグナル伝達に関する分子、レセプターおよびイオンチャネル）の検出のための分子または試薬と結合して、本明細書中で提供されるような表面修飾蛍光半導体微粒子を含む。 Fluorescent semiconductor particulate conjugates also detect biological compounds (eg, enzymes, enzyme substrates, enzyme inhibitors, intracellular organelles, lipids, phospholipids, fatty acids, sterols, cell membranes, signaling molecules, receptors and ion channels). Including surface-modified fluorescent semiconductor microparticles as provided herein in combination with molecules or reagents for
当該結合体はまた、細胞形態および流体の流れ；細胞生存能力、増殖および機能；エンドサイトーシスおよびエキソサイトーシス（Ｂｅｔｚら（１９９６）Ｃｕｒｒ．Ｏｐｉｎ．Ｎｅｕｒｏｂｉｏｌ．６（３）：３６５−７１）；および反応性酸素種（例えば、スーパーオキシド、一酸化窒素、ヒドロキシラジカル、酸素ラジカル）を検出するために使用され得る。さらに、結合体は、生物系の疎水性領域または親水性領域を検出するために使用され得る。 The conjugate also has cell morphology and fluid flow; cell viability, proliferation and function; endocytosis and exocytosis (Betz et al. (1996) Curr. Opin. Neurobiol. 6 (3): 365-71); And can be used to detect reactive oxygen species (eg, superoxide, nitric oxide, hydroxy radicals, oxygen radicals). Furthermore, the conjugate can be used to detect hydrophobic or hydrophilic regions of biological systems.
生体物質蛍光標識剤（蛍光半導体微粒子結合体）はまた、他の多くの生物学的および非生物学的な用途における有用性を見い出し、ここで、発光マーカー、特に蛍光マーカーが代表的に使用される。例えば、Ｈａｕｇｌａｎｄ、Ｒ．Ｐ．Ｈａｎｄｂｏｏｋ ｏｆ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｂｅｓ ａｎｄ Ｒｅｓｅａｒｃｈ Ｃｈｅｍｉｃａｌｓ（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ、Ｅｕｇｅｎｅ、ＯＲ．第６版１９９６；Ｗｅｂｓｉｔｅ，ｗｗｗ．ｐｒｏｂｅｓ．ｃｏｍ．）が参考になる。 Biomaterial fluorescent labeling agents (fluorescent semiconductor particulate conjugates) also find utility in many other biological and non-biological applications, where luminescent markers, particularly fluorescent markers, are typically used. The For example, Haugland, R .; P. Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, OR. 6th Edition 1996; Website, www.probes.com.).
本発明に係る生体物質蛍光標識剤が有用である領域の例としては、蛍光免疫細胞化学、蛍光顕微鏡法、ＤＮＡ配列分析、蛍光インサイチュハイブリダイゼーション（ＦＩＳＨ）、蛍光共鳴エネルギー移動（ＦＲＥＴ）、フローサイトメトリー（蛍光活性化シェルソーター；ＦＡＣＳ）および生物系についての診断アッセイ等が挙げられるが、これらに制限されない。 Examples of areas where the biological material fluorescent labeling agent according to the present invention is useful include fluorescence immunocytochemistry, fluorescence microscopy, DNA sequence analysis, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), flow site Examples include, but are not limited to, measurement (fluorescence activated shell sorter; FACS) and diagnostic assays for biological systems.
上記の領域におけるナノ結晶結合体の有用性に関するさらなる議論については、Ｂａｗｅｎｄｉらに対する国際特許公開ＷＯ ００／１７６４２が参考になる。 For further discussion on the usefulness of nanocrystal conjugates in the above areas, reference is made to International Patent Publication WO 00/17642 to Bawendi et al.
以上、上述のように、本発明の応用範囲は固定細胞を用いた免疫染色、細胞観察やレセプター・リガンド（低分子、薬物）相互作用のリアルタイムトラッキング、１分子蛍光イメージングでなどが挙げられる。 As described above, the application range of the present invention includes immunostaining using fixed cells, cell observation, real-time tracking of receptor-ligand (low molecule, drug) interaction, and single molecule fluorescence imaging.
以下、実施例により本発明をより詳細に説明するが、本発明はこれに限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.
実施例１
（Ｓｉコア粒子及びＳｉ／ＳＩＯ2・コア／シェル粒子の調製）
〈ＨＦエッチング法〉
熱処理したＳｉＯx（ｘ−１．９９９）のフッ酸中溶解によりＳｉの蛍光半導体微粒子（以下において「Ｓｉ半導体微粒子」又は「Ｓｉコア粒子」ともいう。）を製造する場合、先ず、プラズマＣＶＤによりシリコンウエハー上に成膜したＳｉＯx（ｘ−１．９９９）を不活性ガス雰囲気中で１１００℃、１時間程度アニールを行う。これにより、ＳｉＯ2膜中にＳｉ半導体微粒子（結晶）が析出する。Example 1
(Preparation of Si core particles and Si / SIO 2 .core / shell particles)
<HF etching method>
In the case of producing Si fluorescent semiconductor fine particles (hereinafter also referred to as “Si semiconductor fine particles” or “Si core particles”) by dissolving heat-treated SiO x (x-1.999) in hydrofluoric acid, first, plasma CVD is used. SiO x (x-1.999) formed on a silicon wafer is annealed at 1100 ° C. for about 1 hour in an inert gas atmosphere. Thereby, Si semiconductor fine particles (crystals) are precipitated in the SiO 2 film.
次に、このシリコンウエハーを室温で１％程度のフッ酸水溶液で処理することによりＳｉＯ2膜を除去し、液面に凝集した数ｎｍサイズのＳｉ半導体微粒子を回収する。なお、このフッ酸処理により、半導体微粒子（結晶）表面のＳｉ原子のダングリングボンド（未結合手）が水素終端され、Ｓｉ結晶が安定化する。その後、回収したＳｉ半導体微粒子の表面を酸素雰囲気中で自然酸化し、又は加熱して熱酸化し、Ｓｉ半導体微粒子からなるコアの周囲にＳｉＯ2からなるシェル層を形成する。Next, this silicon wafer is treated with a 1% hydrofluoric acid aqueous solution at room temperature to remove the SiO 2 film, and several nanometer-sized Si semiconductor fine particles aggregated on the liquid surface are collected. By this hydrofluoric acid treatment, dangling bonds (unbonded bonds) of Si atoms on the surface of the semiconductor fine particles (crystal) are terminated with hydrogen, and the Si crystal is stabilized. Thereafter, the surface of the collected Si semiconductor fine particles is naturally oxidized in an oxygen atmosphere or is thermally oxidized by heating to form a shell layer made of SiO 2 around the core made of the Si semiconductor fine particles.
〈陽極酸化法〉
また、ｐ型シリコンウエハーの陽極化成によりＳｉ半導体微粒子を製造する場合、先ず、フッ酸（４６％）、メタノール（１００％）及び過酸化水素水（３０％）を１：２：２の割合で混合した溶液中で、ｐ型シリコンウエハー及び白金を対向電極として３２０ｍＡ／ｃｍ2で約１時間通電し、Ｓｉ半導体微粒子（結晶）を析出さシェル。このようにして得られたＳｉ半導体微粒子の表面を酸素雰囲気中で自然酸化し、又は加熱して熱酸化し、Ｓｉ結晶からなるコアの周囲にＳｉＯ2からなるシェル層を形成する。<Anodic oxidation method>
In the case of producing Si semiconductor fine particles by anodizing a p-type silicon wafer, first, hydrofluoric acid (46%), methanol (100%) and hydrogen peroxide solution (30%) are used in a ratio of 1: 2: 2. In the mixed solution, a p-type silicon wafer and platinum were used as a counter electrode and current was applied at 320 mA / cm 2 for about 1 hour to deposit Si semiconductor fine particles (crystals). The surface of the Si semiconductor fine particles thus obtained is naturally oxidized in an oxygen atmosphere or thermally oxidized by heating to form a shell layer made of SiO 2 around the core made of Si crystal.
（Ｓｉ／ＺｎＳ・コア／シェル粒子の調製）
上記で得られたＳｉコア粒子をピリジン中に分散させ１００℃に保温。別途、Ｚｎ（Ｃ2Ｈ5）2と（（ＣＨ3）3Ｓｉ）2Ｓ、Ｐ（Ｃ4Ｈ9）3をアルゴンガス雰囲気下、超音波をかけながらｐＨを制御しながらゆっくり混合した。(Preparation of Si / ZnS / core / shell particles)
The Si core particles obtained above were dispersed in pyridine and kept at 100 ° C. Separately, Zn (C 2 H 5 ) 2 , ((CH 3 ) 3 Si) 2 S, and P (C 4 H 9 ) 3 were slowly mixed in an argon gas atmosphere while controlling the pH while applying ultrasonic waves.
これをピリジン分散液に滴下して添加。添加後、温度を適正に制御し、ｐＨを一定（２５℃において８．５）に保ちゆっくり３０分攪拌した。これの遠心分離を行い沈降した粒子を捕集した。得た粒子の元素分析を行ってみたところＳｉとＺｎＳが確認され、ＸＰＳ分析によりＺｎＳがＳｉの表面に被覆していることがわかった。 This is added dropwise to the pyridine dispersion. After the addition, the temperature was appropriately controlled, the pH was kept constant (8.5 at 25 ° C.), and the mixture was stirred slowly for 30 minutes. This was centrifuged and the settled particles were collected. When elemental analysis of the obtained particles was performed, Si and ZnS were confirmed, and it was found by XPS analysis that ZnS was coated on the surface of Si.
（比較粒子形成１：ＣｄＳｅ／ＳｉＯ2・コア／シェル粒子の調製）
酢酸カドミウム０．１４ｇとトリオクチルフォスフィンオキシド（ＴＯＰＯ）５．０ｇをナスフラスコに入れ、アルゴンで系内を満たした後、所定の温度（１５０〜２５０℃）まで加熱した。この溶液に、２５ｍｇ／ｃｍ3の濃度となるようにセレンを溶解させたトリｎ−オクチルフォスフィン溶液１．４４ｃｍ3を、激しく撹拌しながら素早く注入し、さらに１時間撹拌することによりＴＯＰＯ安定化ＣｄＳｅ（以下、「ＴＯＰＯ／ＣｄＳｅ」と呼ぶ。）を得た。２００℃及び１５０℃でＴＯＰＯ／ＣｄＳｅを合成した場合、ＣｄＳｅ半導体微粒子の吸収スペクトルの立ち上がり波長は、それぞれ、６５０ｎｍと６１０ｎｍであり、その平均粒径は、それぞれ５．５ｎｍと４．５ｎｍであった。このＴＯＰＯ／ＣｄＳｅ粉末を用いて、３−メルカプトプロピルトリメトキシシランでＣｄＳｅ半導体微粒子表面を修飾しさらに加水分解することにより粒子表面にシリカ薄膜を形成させＣｄＳｅ・コア／シリカ・シェル構造体（以下、「ＣｄＳｅ／ＳｉＯ2」と呼ぶ。）を得た。(Comparison particle formation 1: Preparation of CdSe / SiO 2 core / shell particles)
Cadmium acetate (0.14 g) and trioctylphosphine oxide (TOPO) (5.0 g) were placed in a recovery flask, filled with argon, and then heated to a predetermined temperature (150 to 250 ° C.). To this solution, TOPO stabilized by a tri-n- octyl phosphine solution 1.44 cm 3 was dissolved selenium to a concentration of 25 mg / cm 3, quickly injected with vigorous stirring and stirred for a further 1 hour CdSe (hereinafter referred to as “TOPO / CdSe”) was obtained. When TOPO / CdSe was synthesized at 200 ° C. and 150 ° C., the rising wavelengths of the absorption spectra of CdSe semiconductor fine particles were 650 nm and 610 nm, respectively, and the average particle diameters were 5.5 nm and 4.5 nm, respectively. . Using this TOPO / CdSe powder, the surface of the CdSe semiconductor fine particles is modified with 3-mercaptopropyltrimethoxysilane and further hydrolyzed to form a silica thin film on the surface of the particles to form a CdSe-core / silica-shell structure (hereinafter referred to as referred to as the "CdSe / SiO 2".) was obtained.
得られたコア／シェル構造体を光溶解液中で単色光（５６０ｎｍ）を照射することで、コア／シェル構造体内部のセレン化カドミウム（ＣｄＳｅ）半導体微粒子にサイズ選択光エッチングを適用し、セレン化カドミウム（ＣｄＳｅ）半導体微粒子の粒径を約３．５ｎｍにまで減少させた、コア／シェル構造体からなる蛍光半導体微粒子を得た。 The obtained core / shell structure is irradiated with monochromatic light (560 nm) in a photodissolving solution to apply size selective photoetching to cadmium selenide (CdSe) semiconductor fine particles inside the core / shell structure. Fluorescent semiconductor fine particles having a core / shell structure in which the particle size of cadmium iodide (CdSe) fine particles was reduced to about 3.5 nm were obtained.
（比較粒子形成２：ＣｄＳｅ／ＺｎＳ・コア／シェル粒子の調製）
酢酸カドミウム０．１４ｇとトリオクチルフォスフィンオキシド（ＴＯＰＯ）５．０ｇをナスフラスコに入れ、アルゴンで系内を満たした後、所定の温度（１５０〜２５０℃）まで加熱した。この溶液に、２５ｍｇ／ｃｍ3の濃度となるようにセレンを溶解させたトリｎ−オクチルフォスフィン溶液１．４４ｃｍ3を、激しく撹拌しながら素早く注入し、さらに１時間撹拌することによりＴＯＰＯ安定化ＣｄＳｅ（以下、「ＴＯＰＯ／ＣｄＳｅ」と呼ぶ。）を得た。(Comparison particle formation 2: Preparation of CdSe / ZnS / core / shell particles)
Cadmium acetate (0.14 g) and trioctylphosphine oxide (TOPO) (5.0 g) were placed in a recovery flask, filled with argon, and then heated to a predetermined temperature (150 to 250 ° C.). To this solution, TOPO stabilized by a tri-n- octyl phosphine solution 1.44 cm 3 was dissolved selenium to a concentration of 25 mg / cm 3, quickly injected with vigorous stirring and stirred for a further 1 hour CdSe (hereinafter referred to as “TOPO / CdSe”) was obtained.
上記で得られたＣｄＳｅコア粒子をピリジン中に分散させ１００℃に保温。別途、Ｚｎ（Ｃ2Ｈ5）2と（（ＣＨ3）3Ｓｉ）2Ｓ、Ｐ（Ｃ4Ｈ9）3をアルゴンガス雰囲気下、ゆっくり混合した。The CdSe core particles obtained above were dispersed in pyridine and kept at 100 ° C. Separately, Zn (C 2 H 5 ) 2 , ((CH 3 ) 3 Si) 2 S, and P (C 4 H 9 ) 3 were slowly mixed in an argon gas atmosphere.
これをピリジン分散液に滴下して添加。添加後、温度を適正に制御し、ｐＨを一定（２５℃において８．５）に保ちゆっくり３０分攪拌した。これの遠心分離を行い沈降した粒子を捕集した。得た粒子の元素分析を行ってみたところＩｎＰとＺｎＳが確認され、ＸＰＳ分析によりＺｎＳがＣｄＳｅの表面に被覆していることがわかった。 This is added dropwise to the pyridine dispersion. After the addition, the temperature was appropriately controlled, the pH was kept constant (8.5 at 25 ° C.), and the mixture was stirred slowly for 30 minutes. This was centrifuged and the settled particles were collected. When elemental analysis of the obtained particles was performed, InP and ZnS were confirmed, and it was found by XPS analysis that ZnS was coated on the surface of CdSe.
以上のように製造されたコア及びコアシェル粒子の粒径をシスメックス社製：ゼータサイザーＺＳで測定し、比重をメトラー社製：ＳＧＭ−６で測定し、結果を表１に示した。 The particle diameters of the core and core-shell particles produced as described above were measured with Sysmex Corporation: Zetasizer ZS, the specific gravity was measured with Mettler Corporation: SGM-6, and the results are shown in Table 1.
（修飾官能基の導入）
上記蛍光半導体微粒子により生体物質を標識する場合、当該粒子と生体物質のどちらかもしくは双方に、互いに結合する官能基等を導入する必要があるが、下記のように行った。(Introduction of modified functional groups)
When labeling a biological material with the fluorescent semiconductor fine particles, it is necessary to introduce functional groups or the like that are bonded to each other or both of the particle and the biological material.
〈Ｓｉ／ＳｉＯ2・コア／シェル粒子への修飾官能基の導入〉
メルカプト基（ＳＨ基）同士の結合を利用して蛍光半導体微粒子にカルボキシル基を導入する。<Introduction of modified functional groups into Si / SiO 2 core / shell particles>
A carboxyl group is introduced into the fluorescent semiconductor fine particles by utilizing a bond between mercapto groups (SH groups).
先ず、上記のＳｉコア粒子を３０％過酸化水素水中に１０分間分散させ、結晶表面を水酸化さシェル。次に、溶剤をトルエンに置換し、メルカプトプロピルトリエトキシシランをトルエンの２％加えて、２時間程度かけてＳｉコア粒子の最表面のＳｉＯ2をシラン化すると共にメルカプト基を導入する。続いて、溶剤を純水に置換してバッファ塩を添加し、さらに一端にメルカプト基の導入された１１−メルカプトウンデカン酸を適量加えて３時間攪拌することで、Ｓｉコア粒子と１１−メルカプトウンデカン酸とを結合さシェル。これは、本発明においては生体に対し親和結合する修飾基を導入した例である。これを標識Ａとする。First, the above Si core particles are dispersed in 30% hydrogen peroxide water for 10 minutes, and the crystal surface is hydroxylated shell. Next, the solvent is replaced with toluene, and 2% of mercaptopropyltriethoxysilane is added to toluene to silanize SiO 2 on the outermost surface of the Si core particles and introduce a mercapto group over about 2 hours. Subsequently, the solvent is replaced with pure water, a buffer salt is added, and an appropriate amount of 11-mercaptoundecanoic acid having a mercapto group introduced is added to one end, followed by stirring for 3 hours, whereby Si core particles and 11-mercaptoundecane are added. Shell combined with acid. This is an example in which a modifying group capable of affinity binding to a living body is introduced in the present invention. This is labeled A.
〈Ｓｉ／ＺｎＳ・コアシェル粒子への修飾官能基の導入〉
上記で得たＳｉ／ＺｎＳ・コア／シェル粒子をバッファ塩溶液に分散して、１１−メルカプトウンデカン酸を適量加えて適温で２時間攪拌し、粒子表面にメルカプト基を結合させた。これにより表面にカルボキシル基が導入される。これを標識Ｂとする。<Introduction of modified functional groups into Si / ZnS / core shell particles>
The Si / ZnS / core / shell particles obtained above were dispersed in a buffer salt solution, and an appropriate amount of 11-mercaptoundecanoic acid was added and stirred at an appropriate temperature for 2 hours to bond mercapto groups to the particle surfaces. This introduces carboxyl groups on the surface. This is labeled B.
〈ＣｄＳｅ／ＳｉＯ2・コア／シェル粒子への修飾官能基の導入〉
標識Ａと同様な方法で１１−メルカプトウンデカン酸を表面に結合し、カルボキシル基を導入した。標識Ｃとする。<Introduction of modified functional groups into CdSe / SiO 2 core / shell particles>
11-mercaptoundecanoic acid was bound to the surface in the same manner as for label A, and a carboxyl group was introduced. This is labeled C.
〈ＣｄＳｅ／ＺｎＳコアシェル粒子への修飾官能基の導入〉
標識Ｂと同様な方法で１１−メルカプトウンデカン酸を表面に結合し、カルボキシル基を導入した。標識Ｄとする。<Introduction of modified functional groups into CdSe / ZnS core-shell particles>
11-mercaptoundecanoic acid was bound to the surface in the same manner as for label B, and a carboxyl group was introduced. This is labeled D.
＜蛍光強度解析＞
各標識について４０５ｎｍの光を励起光とし、蛍光スペクトロメーターＦＰ−６５００（日本分光）にて蛍光強度を評価した。標識Ａを基準１００として相対値で表し、表１に示した。<Fluorescence intensity analysis>
The fluorescence intensity was evaluated with a fluorescence spectrometer FP-6500 (JASCO) using 405 nm light as excitation light for each label. The label A is expressed as a relative value with reference 100, and is shown in Table 1.
＜細胞への取り込み染色解析＞
上記で得た標識を事前に羊血清アルブミン（ＳＳＡ）と等濃度で混和し、個別にＶｅｒｏ細胞へ取り込ませた。３７℃２時間培養した後、トリプシン処理して５％ＦＢＳ加ＤＭＥＭ再浮遊させ、同一ガラスボトムディッシュに播種した。３７℃で一晩培養した細胞は４％ホルマリンで固定しＤＡＰＩで核を染色して、共焦点レーザースキャン顕微鏡（励起４０５ｎｍ）で蛍光観察を行った。<Cell uptake staining analysis>
The label obtained above was previously mixed with sheep serum albumin (SSA) at an equal concentration, and individually incorporated into Vero cells. After culturing at 37 ° C. for 2 hours, it was treated with trypsin, resuspended in DMEM with 5% FBS, and seeded on the same glass bottom dish. Cells cultured overnight at 37 ° C. were fixed with 4% formalin, nuclei were stained with DAPI, and fluorescence was observed with a confocal laser scanning microscope (excitation 405 nm).
本標識の細胞質のエンドソームへの集積状態を蛍光強度に依存する濃度および分散状態で評価した。即ち本標識が細胞へ取り込まれてエンドソームへ移動集積の移動効率が高い場合はエンドソームでの蛍光強度が高く、その分布面積も広い。一方、粒経および比重などの影響で取り込み、移動率が低い場合には蛍光強度は低く、面積も小さい。この観察の様子を表１に記した。 The state of cytoplasmic endoplasmic accumulation of this label was evaluated at a concentration and dispersion state depending on the fluorescence intensity. That is, when the label is incorporated into cells and the transfer efficiency of the transfer accumulation into the endosome is high, the fluorescence intensity in the endosome is high and the distribution area is wide. On the other hand, the fluorescence intensity is low and the area is small when it is taken in due to the influence of particle size and specific gravity and the migration rate is low. The state of this observation is shown in Table 1.
またテキサスレッドで標識して染色した結果も表１に併せて記した。 The results of labeling and staining with Texas Red are also shown in Table 1.
表１に示した結果からわかるように本発明に係る標識Ａ、Ｂは細胞への取り込み移動度が良く細胞イメージング観察に適していることがわかる。また色素に比べても蛍光強度が高く、耐久性も多角よりイメージングに最適であることも理解できる。 As can be seen from the results shown in Table 1, it can be seen that the labels A and B according to the present invention have good uptake mobility into cells and are suitable for cell imaging observation. It can also be understood that the fluorescence intensity is higher than that of the dye and the durability is optimal for imaging from various aspects.
実施例２
実施例１で調製したコア／シェル粒子を用いてアビジン修飾を行った。Example 2
Avidin modification was performed using the core / shell particles prepared in Example 1.
Ｓｉ／ＳｉＯ2・コア／シェル粒子を濃硫酸と過酸化水素水との３：１混合液中で１０分程反応させ、結晶粒子表面を水酸化さシェル。水洗後、水とエタノールの１：１混合溶液中でアミノプロピルトリエトキシシランを当該粒子の最表面の水酸基と反応させ、粒子にアミノ基を導入する。反応後水洗し、溶媒をアセトンに置換して１ｍｏｌ／ｌの濃度になるようにマレイン酸を加え、３０〜４５分程度、沸点近くまで加熱・還流することで、アミノプロピルエトキシシランのアミノ基とマレイン酸のカルボキシル基を結合さシェル。反応後水洗し、８６９ｍＭのＮ−ヒドロキシスクシンイミド水溶液と、５２２ｍＭのＮ−（３−ジメチルアミノプロピル）−Ｎ’−エチルカルボジイミトーヒドロクロライド水溶液の１：１混合液に分散させ１０分程度反応さシェル。反応後水洗し、室温・水溶液中で２．２１μＭのアビジンと２時間反応させた後、１ｍＭのエタノールアミンを加え、室温で１０分間攪拌する。これにより粒子にアビジンが結合される。これを標識Ａ−２とする。Si / SiO 2 .core / shell particles are reacted in a 3: 1 mixture of concentrated sulfuric acid and hydrogen peroxide for about 10 minutes, and the surface of the crystal particles is hydroxylated shell. After washing with water, aminopropyltriethoxysilane is reacted with a hydroxyl group on the outermost surface of the particles in a 1: 1 mixed solution of water and ethanol to introduce amino groups into the particles. After the reaction, washing with water, replacing the solvent with acetone, adding maleic acid to a concentration of 1 mol / l, heating and refluxing to near the boiling point for about 30 to 45 minutes, the amino group of aminopropylethoxysilane and Shell bonded with the carboxyl group of maleic acid. After the reaction, it was washed with water, dispersed in a 1: 1 mixture of 869 mM N-hydroxysuccinimide aqueous solution and 522 mM N- (3-dimethylaminopropyl) -N′-ethylcarbodiimito hydrochloride aqueous solution, and reacted for about 10 minutes. shell. After the reaction, it is washed with water, reacted with 2.21 μM avidin for 2 hours in an aqueous solution at room temperature, 1 mM ethanolamine is added, and the mixture is stirred at room temperature for 10 minutes. This binds avidin to the particles. This is labeled A-2.
〈Ｓｉ／ＺｎＳ・コア／シェル粒子へのアビジンの導入〉
３−メルカプト１−アミノプロパンを用いてメルカプト基で粒子に結合さえて表面にアミノ基を導入した後、上記標識Ａ−２と同様に行い、アビジン結合標識Ｂ−２を得た。<Introduction of avidin into Si / ZnS / core / shell particles>
After 3-mercapto 1-aminopropane was used to bind to the particle with a mercapto group and an amino group was introduced to the surface, the same procedure as in Label A-2 was performed to obtain avidin-bound label B-2.
〈ＣｄＳｅ／ＳｉＯ2・コア／シェル粒子へのアビジンの導入〉
上記標識Ａ−２と同様にしてアビジン結合標識Ｃ−２を得た。<Introduction of avidin into CdSe / SiO 2 core / shell particles>
Avidin-bound label C-2 was obtained in the same manner as label A-2.
〈ＣｄＳｅ／ＺｎＳ・コア／シェル粒子へのアビジンの導入〉
上記標識Ｂ−２と同様にしてアビジン結合標識Ｄ−２を得た。<Introduction of avidin into CdSe / ZnS / core / shell particles>
Avidin-binding label D-2 was obtained in the same manner as label B-2.
＜免疫染色＞
核膜孔輸送を担うＧＴＰ分解酵素Ｐａｎを上記各標識で染色を試みた。ＧＴＰ分解酵素Ｐａｎと標識とを混和しＶｅｒｏ細胞に取り込ませた。３７℃２時間培養した後共焦点レーザースキャン顕微鏡（励起４０５ｎｍ）で蛍光観察を行った。核の染色の状態を評価し表２に記した。観察された蛍光強度が高く、面積が大きければ標識コンジュゲートの移動効率は高く、本発明標識の特長を表すものである。染色がほとんど観察されない場合は比重が高すぎるために沈降・沈降するなどの理由で移動できなかったことが示す。<Immunostaining>
An attempt was made to stain GTP-degrading enzyme Pan, which is responsible for nuclear pore transport, with each of the above labels. GTP-degrading enzyme Pan and a label were mixed and incorporated into Vero cells. After culturing at 37 ° C. for 2 hours, fluorescence observation was performed with a confocal laser scanning microscope (excitation 405 nm). The state of nuclear staining was evaluated and listed in Table 2. When the observed fluorescence intensity is high and the area is large, the transfer efficiency of the labeled conjugate is high, which represents the feature of the label of the present invention. When the staining is hardly observed, the specific gravity is too high, indicating that it cannot move due to sedimentation or sedimentation.
表２に示すように本発明に基づく標識は動態を伴うイメージングに有効であることが分かる。すなわち、生体物質の動態イメージングの描写性を大きく向上できることが分かる。 As shown in Table 2, it can be seen that the label according to the present invention is effective for imaging involving kinetics. That is, it can be seen that the descriptiveness of dynamic imaging of biological materials can be greatly improved.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002038145A (en) * | 2000-07-27 | 2002-02-06 | Mitsubishi Chemicals Corp | Semiconductor superfine particle binding amino group |
| WO2004096944A1 (en) * | 2003-04-30 | 2004-11-11 | Nanosolutions Gmbh | Core/shell nanoparticles suitable for (f)ret-assays |
| JP2006070249A (en) * | 2004-08-03 | 2006-03-16 | Fuji Photo Film Co Ltd | Phosphor particle and fluorescence-detecting method |
| WO2006033396A1 (en) * | 2004-09-22 | 2006-03-30 | Japan Science And Technology Agency | Water-soluble fluorescent material and method for producing same |
| WO2006060041A1 (en) * | 2004-08-04 | 2006-06-08 | Agency For Science, Technology, And Research | Coated water soluble nanoparticles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH074243B2 (en) * | 1986-11-04 | 1995-01-25 | 日東電工株式会社 | Carrier particles for immobilizing redispersible bioactive substances |
| JPH05281230A (en) * | 1991-04-08 | 1993-10-29 | Toyobo Co Ltd | Method for highly sensitive detection of ligand-receptor reaction |
| JPH06279015A (en) | 1993-03-30 | 1994-10-04 | Matsushita Electric Ind Co Ltd | Production of ultrafine silicon particle |
| JPH10310770A (en) | 1997-03-10 | 1998-11-24 | Sony Corp | Production of luminophor |
| CA2344478C (en) | 1998-09-18 | 2010-03-30 | Massachusetts Institute Of Technology | Biological applications of semiconductor nanocrystals |
| CA2345376C (en) * | 1998-09-24 | 2010-03-16 | Advanced Research And Technology Institute, Inc. | Water-soluble luminescent quantum dots and bioconjugates thereof |
| JP2000104058A (en) | 1998-09-28 | 2000-04-11 | Sony Corp | Production of luminescent material |
| US6585947B1 (en) | 1999-10-22 | 2003-07-01 | The Board Of Trustess Of The University Of Illinois | Method for producing silicon nanoparticles |
| US6179912B1 (en) * | 1999-12-20 | 2001-01-30 | Biocrystal Ltd. | Continuous flow process for production of semiconductor nanocrystals |
| JP2001249131A (en) * | 2000-03-07 | 2001-09-14 | Mitsubishi Chemicals Corp | Labeling particle and method of manufacturing it |
| JP2002139495A (en) * | 2000-11-06 | 2002-05-17 | Kasei Optonix Co Ltd | Phosphor bead for tracer analysis and its producing method |
| EP1241713A1 (en) | 2001-02-07 | 2002-09-18 | Agfa-Gevaert | Preparation of improved ZnS: Mn phosphors |
| ATE448077T1 (en) * | 2001-09-17 | 2009-11-15 | Life Technologies Corp | NANOCRYSTALS |
| US7205048B2 (en) * | 2001-09-17 | 2007-04-17 | Invitrogen Corporation | Functionalized fluorescent nanocrystal compositions and methods of making |
| JP4107873B2 (en) | 2002-05-09 | 2008-06-25 | 賢一 花木 | Luminescent fine particles |
| JP4245415B2 (en) * | 2002-11-25 | 2009-03-25 | 健二 山本 | Method for producing molecular recognition phosphor |
| JP2004356163A (en) | 2003-05-27 | 2004-12-16 | Toyota Central Res & Dev Lab Inc | Silicon-based thin film, method of manufacturing the same and photoelectric conversion element |
| CN100558769C (en) * | 2003-06-09 | 2009-11-11 | 3M创新有限公司 | A kind of polymer composition that comprises the melt-processable of fluoropolymer with long-chain branch |
| JP4483279B2 (en) * | 2003-12-05 | 2010-06-16 | ソニー株式会社 | Biological substance fluorescent labeling agent, biological substance fluorescent labeling method, and bioassay method and apparatus |
| JP2005239775A (en) | 2004-02-24 | 2005-09-08 | Doshisha | Phosphor and manufacturing method therefor |
| EP1753611A4 (en) * | 2004-05-10 | 2011-09-14 | Evident Technologies | Iii-v semiconductor nanocrystal complexes and methods of making same |
| US7261940B2 (en) * | 2004-12-03 | 2007-08-28 | Los Alamos National Security, Llc | Multifunctional nanocrystals |
-
2007
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002038145A (en) * | 2000-07-27 | 2002-02-06 | Mitsubishi Chemicals Corp | Semiconductor superfine particle binding amino group |
| WO2004096944A1 (en) * | 2003-04-30 | 2004-11-11 | Nanosolutions Gmbh | Core/shell nanoparticles suitable for (f)ret-assays |
| JP2006070249A (en) * | 2004-08-03 | 2006-03-16 | Fuji Photo Film Co Ltd | Phosphor particle and fluorescence-detecting method |
| WO2006060041A1 (en) * | 2004-08-04 | 2006-06-08 | Agency For Science, Technology, And Research | Coated water soluble nanoparticles |
| WO2006033396A1 (en) * | 2004-09-22 | 2006-03-30 | Japan Science And Technology Agency | Water-soluble fluorescent material and method for producing same |
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